The Microbiome Structure of a Rice-Crayfish Integrated Breeding Model and Its Association with Crayfish Growth and Water Quality

ABSTRACT The rice-crayfish (RC) integrated breeding model is an important and special agricultural ecosystem that provides a unique ecological environment for exploring the microbial diversity, composition, and functional capacity. To date, little is known about the effect of the breeding model on microbiome assembly and breeding model-specific microbiome composition and the association of the microbiome with water quality and crayfish growth. In the present study, we assessed the taxonomic shifts in gut and water microbiomes and their associations with water quality and crayfish growth in the RC and crayfish monoculture (CM) breeding models across six time points of rice growth, including seedling (a), tillering and jointing (b), blooming (c), filling (d), fruiting (e), and rotting of rice residues (f). Dominant bacterial phyla, such as Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, were detected in both gut and water microbiomes across breeding models. Notably, the diversity and structure of the gut and water microbiomes significantly (P < 0.001) differed between the RC and CM models, with higher microbial diversity being noted in the RC model than in the CM model. The taxa enriched in the RC model included Bacillus sp., Streptomyces sp., Lactobacillus sp., Prevotella sp., Rhodobacter sp., Bifidobacterium sp., Akkermansia sp., and Lactococcus sp., some of which are potentially beneficial to animals. However, opportunistic pathogens, such as Citrobacter sp. and Aeromonas sp., were depleted in the RC model. Furthermore, in the RC model, the enriched taxa that formed complex cooccurrence networks showed a significant positive correlation with water quality and crayfish growth, whereas the depleted taxa showed a significant negative correlation with water quality and crayfish growth. These results suggest that the RC model has a better microbiome composition and that RC model-specific microbes could play important roles in improving crayfish growth and water quality. IMPORTANCE The present study comprehensively compared two different breeding models in terms of their microbiome composition and the associations of the microbiomes with crayfish growth and water quality. RC model-specific microbiome composition was identified; these microbes were found to have a positive association with water quality and crayfish growth. These results provide valuable information for guiding microbial isolation and culture and for potentially harnessing the power of the microbiome to improve crayfish production and health and water quality.

In the present study, the authors assessed the taxonomic shifts in gut and water microbiomes and their associations with water quality and crayfish growth in RC and crayfish monoculture (CM) breeding models. The result was sound in some content, that could supply some valuable information to the literature and aquaculture practice. However, there are numerous concerns to address, especially the experimental design and grammatical assignments.
Major points 1. in the experiment design, the author hypothesize that breeding model-specific microbes have significant associations with water quality and crayfish growth. However, the dietary pattern and sediment microbes are totally different between these two models. Therefore, it is not entirely reasonable to conclude the microbes in the crayfish gut was only associated with that in the water. To clearly illustrate the crayfish growth associated microbes between these two breeding models, the authors should at least include the microbe alteration from the sediment during the experiment. Fig 7, the samples collected are not representative. In detail, the crayfish determined in stage a is larger in size than the other growth stages, this is not illogical or the actual growth of animals. Meanwhile, the crayfish in c stage under RC model is smaller than that in b stage; TL and AbL between b and c growth stage under CM condition showed opposite trends with other parameters.

In
3. Line 234-238, to be continued with the last comments, if the growth performance is not illogical under different stages, the correlation between crayfish growth and indicator OTUs in the water and gut microbiome is not illogical. 4. in discussion section, the authors expounded an ambiguous description on how the RC model shaped the gut microbes. In addition, the relationship between water and gut microbes, as well as their action mechanisms on crayfish growth should be well interpreted.
Minor points This manuscript should be carefully review by a native English speaker to make it easier read. Staff Comments:

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Introduction
Lines 85-95, Please, check the whole sentence, it is repited twice.

Results
Lines 100-103, please re-write the paragraph: How many water and gut samples from the RC and how many water and gut samples from the MC from three landfields?. Please add a description of the letters a-f described in figure 1.
Line 108, what was the criteria used to first collect samples individually and then pooled to obtain the microbiome. Please, note that there are several differences during seasons and locations.
Line 111. Please add the developmental stages analyzed in this study. Add an additional column in the supplementary table S2 and Table 2B with the number of organisms by developmental stage in RC and MC.
Line 115. Please check this analysis, t student is a parametric test. Is alpha diversity a parametric variable?. The authors need to be aware that are comparing gut microbiota across several developmental stages (from the same period of sampling?, from different periods of sampling?. This comment is also for the water microbiota.
Line 153. Add "sp" after the description of each genus. Line 252. Fiber content was not measured in this study.
Line 256. In the M & M section it was mentioned that the gut microbiome was not done.

Discussion
Lines 240-242. Delete "we compared the crayfish gut and water microbiomes between two different breeding models in order to better understand breeding model-specific microbes and their relationship with water quality and crayfish growth".
Line 247. The diversity and community structure of both gut and water microbiome was different between RC and MC". Date of harvesting would have contributed to this. In none of the analyses the authors included time. Nutrients were important but what other abiotic parameters would have contributed?, sediments were also important?.
Line 254-256. This paragraph is repeated. Delete it. "Consistent with this finding, the bacterial diversity in both water and gut microbiomes was found to be higher in the RC model than in the CM model in the present study".
Line 260-263. This paragraph is quite speculative. The authors did not evaluate the presence of fiber or lignin.
Line 295-299. Basing the quality of the water only in the concentration of ammonia, nitrite and phosphorus contents is quite limited. Please discuss fully what other variables are missing?

Material and methods
Line 344. Please describe the stocking density in both RC and CM models?. Did the organisms were fed with artificial food?. Comment: Figure 1a-f is describing the characteristics of each landscape either for water or gut microbiota collection. The authors need to describe fully the experimental design.
Please also describe the criteria used for sampling collection?, is it from the surface?, the middle? or bottom water colum?.
For the water collection, the authors collected 2 l from 5 sampling points and pooled them into 10 L. then, they collected 500 ml for the biota collection. What happed with the remained 9,500 ml?. What was the criteria to follow this procedure?.
Line 347. Please describe the larval stages evaluated?. Did they evaluated crayfish growth during rice harvesting or after?.
Line 348. How did the authors assessed the growth of crayfish?, did they used recaptured methods?.
Line 349, Please describe fully the dissection process and add a reference for this methodology.
Line 356: Please, re-describe the whole paragraph for RC and MC. The authors collected 8 organisms from 5 sampling sites. That means 40 organisms x 3 cultured areas?.
Line 357. Please describe fully the stage of each crayfish. By reading the paragraph I can assume that the authors analyzed the gut microbiome of crayfish from June to October, resembling the whole cycle of crayfish?. Please describe fully the process of sampling collections with dates and stages of crayfish collections and also if mature and immature crayfishes were harvested.
Line 377. Please describe the reason for using the V4 region in this study. What is the advantage of using the V4 region vs the 16S rDNA V3-V4 region?. Line 384. How did the authors solve the "chimeric sequences"? Line 391.What was the sequencing depth (in reads) to rarefied the featured

Responds to the reviewer's comments:
We really appreciate your professional and constructive suggestions. We have studied your comments carefully and have made revisions which were highlight by yellow color in the revised manuscript. Besides, we consulted professional English editor to improve our writing, and the editorial certificate was attached to the end of this letter.
The followings are our responses to your comments point by point.

Editor:
Please include in your discussion a brief comparison of your findings compared to those from the following paper: Wang  Response: Thank you for your good suggestions. We have added a comparison of our results compared to the paper "Microbiome Analysis Reveals Microecological Balance in the Emerging Rice-Crayfish Integrated Breeding Mode" in the discussion section as follows. "In the present study, we found that several bacterial phyla, such as Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, were dominant in both gut and water microbiomes of two breeding models across multi-time points; these findings are similar to previous findings in microbiomes of rearing water and guts of other crustaceans (16-20). Compared to the CM model, the diversity and community structure of both gut and water microbiomes were significantly differed in the RC model, suggesting that the breeding model may affect the microbiome assembly of crayfish gut and water. However, these findings were not consistent with the previous study, in which water, sediment, and crayfish samples from single time point were collected, and no statistically significant differences of microbiome diversity and structure between the RC and CM models were found (19). Due to the different environment status of the RC model at different times, we supposed that the sampling time could be a reasonable factor cause of the different results. Amounts of studies have reported a variable microbiome structure at different sampling times (21-23).
Also, our results showed a fluctuating diversity and structure of both water and gut microbiomes across six sampling times. Therefore, we used multi-time points sampling strategy to comprehensively capture the microbiome for the RC and CM models (Page 10, lines 234-250).

Introduction
Lines 85-95, Please, check the whole sentence, it is repeated twice.
Response: Thank you for your good suggestion, we rewrote this paragraph to make our hypothesis, approaches, and aims clearer (Page 4, lines 85-96).
Response: Thank you for your good suggestion, we rewrote this paragraph to make our hypothesis, approaches, and aims clearer (Page 4, lines 85-96).

3, Response to comment: Results
Lines 100-103, please re-write the paragraph: How many water and gut samples from the RC and how many water and gut samples from the MC from three land fields?
Please add a description of the letters a-f described in figure 1. Line 108, what was the criteria used to first collect samples individually and then pooled to obtain the microbiome. Please, note that there are several differences during seasons and locations.

Response:
There are many studies have been suggested that microbiome was dynamic across time. Consist with this, there are several differences during seasons you mentioned. To make samples in our study were much more representative and comprehensive of two breeding models, we collected samples across multi-time points, which should be better than the sample of single time point. Furthermore, to make our study was much focused and easily understand, we mainly focused on the comparison between breeding models, but not among different time points. Therefore, we focused on the description of the microbiome structure between two breeding models (pooled all the samples from multi-time point for each model, and each time point between two models).
Line 111. Please add the developmental stages analyzed in this study. Add an additional column in the supplementary table S2 and Table 2B with the number of organisms by developmental stage in RC and MC.
Response: Thank you for your great suggestion. However, to make our study much focused and easy understanding, we mainly focused on the comparison between breeding models, but not among different time points. Therefore, we focused on the description of the microbiome structure between two breeding models. In addition, the time points were related to rice growth but not the crayfish developmental stages.
For all the time points, we only measured the mature crayfish. In the next step, we will focus on the microbiome study across developmental stages of crayfish in two breeding models.
Line 115. Please check this analysis, t student is a parametric test. Is alpha diversity a parametric variable? The authors need to be aware that are comparing gut microbiota across several developmental stages (from the same period of sampling? from different periods of sampling? This comment is also for the water microbiota. Response: Thanks for your critical suggestions. We have replaced the methods of t-student to paired Wilcoxon rank-sum test as to satisfy the data of both gut microbiome and water microbiome. To rule out the differences contributed by time points, we used the paired statistical test (comparison between two breeding models from the same period of sampling) to do the comparison between two breeding models for both gut and water microbiomes.
Response: Thank you for your good suggestion, we have added the "sp." for each genus.
Line 153. Add "sp" after the description of each genus.
Response: Thank you for your good suggestion, we have added the "sp." for each genus. Response: Thank you for your good suggestion, we have removed this content.

Discussion
Line 252. Fiber content was not measured in this study.
Response: Thank you for your comments. We do not measure fiber content in this study due to funding limitation. However, serval previous studies have reported higher fiber content in the RC model than in the CM model. Here, we discussed the possible link between fiber content and microbes in the RC model. Some members of Actinobacteria that contribute significantly to the turnover of complex biopolymers and decomposition of organic matter had higher abundances in the RC model than in the CM model. Moreover, gut microbes positively correlated with fiber intake, such as Ruminococcaceae sp., Bifidobacteria sp., and lactic acid bacteria, were more abundant in the RC model than in the CM model. To confirm those links between fiber content and microbes in the RC model, we need conduct much more experiments, such as fiber content measure, culture of target microbes, and synthetic communities in the next step. Response: Thanks for your critical suggestions. To rule out the differences contributed by time points, we used the paired statistical test (comparison between two breeding models from the same period of sampling) to do the comparison between two breeding models for both gut and water microbiomes. To make our study much focused and easily understand, we mainly focused on the comparison between breeding models, but not among different time points. We agreed with you that many abiotic parameters, such as sediments would contribute the microbiome differences between two breeding models. Due to funding limitation and to make our study much focused and easy understanding, we do not collect the microbial and physicochemical data of sediments in this study. To confirm causative roles between abiotic factors, such as nutrient and sediment, and microbiome in the RC model, the further studies, including abiotic factor measure, culture of target microbes, and synthetic communities need to be conducted in the future.
Line 254-256. This paragraph is repeated. Delete it. "Consistent with this finding, the bacterial diversity in both water and gut microbiomes was found to be higher in the RC model than in the CM model in the present study".
Response: Thank you for your good suggestions. We have revised it. Response: Thanks for your good suggestion. In this study, we measured some of the most important water quality parameters, including water temperature, pH, ammonia, nitrite, nitrogen, and phosphorus due to funding limitation. We found that the concentrations of ammonia, nitrite and phosphorus were different between two breeding model. Therefore, we did the discussion regarding the possible links between microbiome and these water quality parameters. Following your suggestion, we discussed the RC-enriched microbes, which can modulate other water quality parameters, including transparency, total dissolved solids, pH, conductivity, chemical oxygen demand, dissolved oxygen, biological oxygen demand, alkalinity, and hardness. In the future, we need to collect water quality parameters that missed in this study and target microbes to further confirm our findings.

Material and methods
Line 344. Please describe the stocking density in both RC and CM models?
Response: Thank you for your suggestion. We added the stocking density information in the M&M part. For the first season of crayfish cultivation (March-June), about 300 kg/ha crayfish larvae were added in both the RC and CM models at the beginning.
During the first season, the management process, such as stocking density and feeding trial were similar between these two breeding models. For the second season of crayfish cultivation (June-October), no larva was added in the RC model due to crayfish self-propagated inside the paddies, while about 225 kg/ha larvae were added in the CM model at the beginning. The stocking density of crayfish was controlled by removing or adding crayfishes both in RC and CM models for all the season.
Did the organisms were fed with artificial food?
Response: Yes, in the first season, commercial feeds were feeding once a day for both models, while commercial feeds and soybean were added twice a week in the second season for both models. Please also describe the criteria used for sampling collection? is it from the surface? the middle? or bottom water column? For the water collection, the authors collected from 5 sampling points and pooled them into 10 L. then, they collected 500 ml for the biota collection. What happed with the remained 9,500 ml? What was the criteria to follow this procedure?
Response: Thank you for your comments. To increase the representativeness of each pond, a total of 10 L water was collected from 5 sampling spots (surface, middle, and bottom for each sample site). Then we pre-filtered with 100-μm nylon mesh to reduce impurities. After that, 500 ml of them were used for the biota collection, 500 mL of them were used for determining water physicochemical parameters, and the remained 9,000 ml were discarded.
Line 347. Please describe the larval stages evaluated? Did they evaluated crayfish growth during rice harvesting or after?
Response: Thank you for your suggestions. In this study, we only collected the mature crayfishes and measured their growth-related data at each time point. The time points were related to rice growth but the crayfish developmental stages. To make our study much focused and easily understand, we mainly focused on the comparison between breeding models, but not among different time points. In the next step, we will focus on the microbiome study across developmental stages of crayfish in two breeding models.
Line 348. How did the authors assessed the growth of crayfish? did they used recaptured methods?
Response: Yes, we do used recaptured methods. Crayfishes were randomly recaptured, and eight healthy mature crayfishes were selected and pooled into two technical replicates for each pond/field across multi-time points. To assess crayfish growth, we determined and recorded the phenotype of each crayfish, including the total length (TL), total weight (TW), abdominal length (AbL), abdominal weight (AbW), and carapace length (CL) of each crayfish (Table S11)  Line 349, Please describe fully the dissection process and add a reference for this methodology.
Response: Thanks for your good suggestion. We re-described the whole dissection process and added a reference for this methodology. The dissection process was described as follows: "After recorded the phenotype of each crayfish, we collected the gut microbes according to the reference (52). All crayfish dissections were performed under sterile conditions. Surgical tools were sterilized using 75% ethanol and flamed prior to use and between incisions. Sterilizing the body surface of each crayfish with 75% ethanol 3 times. Then the guts of these crayfishes were collected and pooled into sterile 10 mL centrifuge tubes with labels. Next, these guts samples were ground in a sterile grinder to collect gut microbes, and divided into two technical replicates. All "Crayfishes were randomly recaptured, and eight healthy mature crayfishes were selected and pooled into two technical replicates for each pond/field across multi-time points. To assess crayfish growth, we determined and recorded the phenotype of each crayfish, including the total length (TL), total weight (TW), abdominal length (AbL), abdominal weight (AbW), and carapace length (CL) (Table S11). After recorded the phenotype of each crayfish, we collected the gut microbes according to the reference.
All crayfish dissections were performed under sterile conditions. Surgical tools were sterilized using 75% ethanol and flamed prior to use and between incisions.
Sterilizing the body surface of each crayfish with 75% ethanol 3 times. Then the guts of these crayfishes were collected and pooled into sterile 10 mL centrifuge tubes with labels. Next, these guts samples were ground in a sterile grinder to collect gut microbes. All the samples were immediately stored at −80°C until DNA extraction. In total, we obtained 36 crayfish gut samples from 3 RC ponds and 30 crayfish gut    Reviewer #2: 1. In the experiment design, the author hypothesize that breeding model specific microbes have significant associations with water quality and crayfish growth.
However, the dietary pattern and sediment microbes are totally different between these two models. Therefore, it is not entirely reasonable to conclude the microbes in the crayfish gut was only associated with that in the water. To clearly illustrate the crayfish growth associated microbes between these two breeding models, the authors should at least include the microbe alteration from the sediment during the experiment Response: Thank you for your comments. We agreed with you that dietary pattern and sediment composition were different between these two models due to the rice planting in RC model. The dietary pattern difference between two models have been confirmed in previous study "Feeding habits of Procambarus clarkii and food web structure in two different aquaculture system" (in Chinese with English abstract, Doi: 10.7541/2020.016). The sediment composition could be different due to the rotting of rice residues and application of artificial fertilizer. In current study, we also found the growth data of crayfish and physicochemical data of water were different between these two models. Based on those differences, we compared the microbiome composition of crayfish gut and water between two models, and generated the breeding model specific microbes. We then found that some of those breeding model specific microbes of crayfish gut and water showed strong association with crayfish growth and water physicochemical features, respectively. In current study, we did not explore the association between gut microbes and water microbes, which has been To make sure the statistical power and reliability of correlation, we collected the data across multi-time points to assess the correlations of crayfish gut microbes with crayfish growth data.
4. in discussion section, the authors expounded an ambiguous description on how the RC model shaped the gut microbes. In addition, the relationship between water and gut microbes, as well as their action mechanisms on crayfish growth should be well interpreted.
Response: Thank you for your comments. In this study, we mainly focused on the comparison of gut and water microbiomes between two models, and the breeding model specific microbes and their association with crayfish growth and water quality.
The relationship of the microbes between the crayfish gut and water in these two breeding models was interesting but has been studied in previous study "Microbiome Analysis Reveals Microecological Balance in the Emerging Rice-Crayfish Integrated Breeding Mode" (Doi: 10.3389/fmicb.2021.669570). We have discussed the possible factors, such as nutrient, nitrogen, and sediment for shaping the gut and water microbiomes. In addition, the relationship between water physiochemical data and water microbiome, as well as the relationship between crayfish growth and gut microbiome were interpreted in discussion section.
5. This manuscript should be carefully review by a native English speaker to make it easier read.
Response: Thank you for your great suggestion. We consulted professional English editor to improve our manuscript, and the editorial certificate was attached to the end of this letter.  Re: Spectrum02204-21R1 (The microbiome structure of a rice-crayfish integrated breeding model and its association with crayfish growth and water quality) Dear Dr. Zemao Gu: Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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